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Structural explanation for the role of Mn2+ in the activity of phi 6 RNA-dependent RNA polymerase

DOI: 10.1093/nar/gkn632 DOI Help

Authors: Minna M. Poranen (Institute of Biotechnology and Department of Biological and Environmental Sciences, University of Helsinki) , Paula Salgado (Imperial College London) , Minni R. L. Koivunen (Institute of Biotechnology and Department of Biological and Environmental Sciences, University of Helsinki) , Sam Wright (University of Oxford) , Dennis H. Bamford (Institute of Biotechnology and Department of Biological and Environmental Sciences, University of Helsinki) , David I. Stuart (Oxford University) , Jonathan M. Grimes (Division of Structural Biology, University of Oxford)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Nucleic Acids Research , VOL 36 (20) , PAGES 6633-6644

State: Published (Approved)
Published: October 2008

Abstract: The biological role of manganese (Mn2+) has been a long-standing puzzle, since at low concentrations it activates several polymerases whilst at higher concentrations it inhibits. Viral RNA polymerases possess a common architecture, reminiscent of a closed right hand. The RNA-dependent RNA polymerase (RdRp) of bacteriophage {phi}6 is one of the best understood examples of this important class of polymerases. We have probed the role of Mn2+ by biochemical, biophysical and structural analyses of the wild-type enzyme and of a mutant form with an altered Mn2+-binding site (E491 to Q). The E491Q mutant has much reduced affinity for Mn2+, reduced RNA binding and a compromised elongation rate. Loss of Mn2+ binding structurally stabilizes the enzyme. These data and a re-examination of the structures of other viral RNA polymerases clarify the role of manganese in the activation of polymerization: Mn2+ coordination of a catalytic aspartate is necessary to allow the active site to properly engage with the triphosphates of the incoming NTPs. The structural flexibility caused by Mn2+ is also important for the enzyme dynamics, explaining the requirement for manganese throughout RNA polymerization.

Subject Areas: Biology and Bio-materials

Facility: Daresbury